Heart failure (HF) is a leading cause of cardiovascular mortality and morbidity in the United States. Despite current treatments, patients with HF suffer from a poor quality of life and reduced lifespan. An improved understanding of the critical pathological mechanisms of HF is required for the development of novel therapies. Hydrogen sulfide (H2S) is a potent endogenous, gaseous signaling molecule that critically regulates cardiovascular homeostasis. H2S regulates blood pressure, inhibits apoptosis and inflammation, protects mitochondria, and exerts powerful antioxidant actions. Previous work from our group has shown that exogenously administered H2S produces robust cardioprotective effects in animal models of heart failure. We have shown that gene-targeted mice that overexpress endogenous H2S producing enzymes are protected in the setting of HF. H2S is generated endogenously by three enzymes cystathionine ?-lyase (CSE), cystathionine ?-synthase (CBS) and 3-mercaptopyruvate sulfurtranseferase (3-MST). CSE, CBS and 3-MST are all expressed in the heart and circulation, but exhibit significant differences in their regulation and cellular localization. Our Central Hypothesis for the proposed studies is that H2S derived from different enzymes, in different cell populations (endothelial cells, cardiac myocytes, fibroblasts) exerts distinct cardioprotective effects in the pathogenesis of HF. Although, we have demonstrated that H2S levels are reduced in the heart and circulation of both laboratory animals and patients with heart failure, the causes and consequences of reduced H2S availability are poorly characterized. We have developed novel gain and loss of function mouse models that will provide mechanistic insights regarding the contribution of CSE, CBS and 3-MST to HF development and progression. We will employ a multifaceted approach that includes physiological, molecular, biochemical, genetic, and pharmacological approaches to elucidate the role of endogenous H2S in heart failure. The proposed studies will evaluate left ventricular structure and function, cardiac fibrosis, exercise capacity, vascular function, mitochondrial bioenergetics, and molecular signaling to evaluate the role of endogenous H2S on the pathobiology of HF. Specifically, we will: (1) determine the time course of expression of all three endogenous H2S generating enzymes as well as the levels of H2S bioavailability in pressure overload and myocardial infarction induced HF; (2) directly investigate the contribution of H2S-producing enzymes in the development and progression of HF pathology through the use of cell type-specific gene-targeted mouse models with gain and loss of function for CSE, CBS, and 3-MST; (3) identify novel endogenous cytoprotective signal cascades mediated via endogenous H2S producing enzymes in the early and late stages of pressure overload and MI induced HF. Successful completion of these studies will further our understanding of the pathogenesis of HF and will provide critical information required for the development of improved pharmacological strategies to harness H2S therapy for the benefit of patients with HF.